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EUROPEAN COMMISSIONHEALTH & CONSUMER PROTECTION DIRECTORATE-GENERAL

Directorate B - Scientific Health OpinionsUnit B3 - Management of scientific committees II

Scientific Committee on FoodSCF/CS/EDUL/192 final

13 March 2000

REVISED OPINION ON CYCLAMIC ACID

AND ITS SODIUM AND CALCIUM SALTS

(Expressed on 9 March 2000)

Rue de la Loi 200, B-1049 Bruxelles/Wetstraat 200, B-1049 Brussel - Belgium - Office: BE232 - 6/37.

Telephone: direct line (+32-2) 295.81.10 / 296.48.70, exchange 299.11.11. Fax: (+32-2) 299.48.91Telex: COMEU B 21877. Telegraphic address: COMEUR Brussels.

http://www.europa.eu.int/comm/dg24/health/sc/scf/index_en.html



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SCF/CS/EDUL/192 final

Revised opinion on cyclamic acid and its sodium and calcium salts

(Expressed on 9 March 2000)

Terms of reference

To review and revise the opinion on cyclamate, expressed on 14th

December 1995, at the 99th

SCF meeting, in the light of scientific information requested in that opinion, now made

available.

This new information consists of the following:

- study on conversion rates in converters and non-converters;

- discussion of in vitro studies to compare testicular sensitivity;- epidemiology studies: intake survey in Catalonia, fertility of workers involved in

cyclamate manufacture, cyclamate intake and male fertility case control study.

Background

The Scientific Committee for Food (SCF) reviewed the toxicity of cyclamate,

cyclohexylamine and dicyclohexylamine in 1985 and established a temporary ADI of 0-11

mg/kg bodyweight (bw), expressed as cyclamic acid, for cyclamic acid and its sodium and

calcium salts (1). The ADI was based on a no-observed-adverse-effect-level (NOAEL) of 100mg/kg bw in the rat for testicular toxicity of cyclohexylamine, the metabolite of cyclamate

produced by microbial fermentation of unabsorbed cyclamate in the lower gut. At that time,the fraction of ingested cyclamate not absorbed was assumed to be 63% and the conversion

rate of unabsorbed cyclamate to cyclohexylamine was estimated to be 30%, giving an overall

conversion rate of 18.9%. The ADI was temporary because of uncertainties relating to the

relevance for man of the testicular damage found in rats fed cyclohexylamine.

The Committee reviewed cyclamate again in 1988, 1991 and 1995 (2-4). The Committee was

informed about studies confirming that cyclohexylamine is metabolised similarly by rat and

man. Further information concerning the intra-individual variability in cyclamate conversionin humans with time and two studies in Cynomolgus monkeys on the testicular toxicity of

cyclohexylamine were submitted by the petitioner. In 1995, preliminary data from three

different epidemiology studies being performed in Spain were also available to the

Committee.

The Committee agreed that these submissions from the petitioner did not answer its questions

about the intra-individual variability of cyclamate conversion with time and the testicular

toxicity of cyclohexylamine. In particular, the monkey studies did not provide any basis for

concluding that primates are less sensitive than the rat to the effects of cyclohexylamine on

the testis. Rather, they had provided evidence that the effects were of toxicological concern inprimates as they were in all other species so far tested with the exception of the mouse.

However, it was not possible to establish a clear NOAEL from the monkey studies. After

considering all new data available, the Committee concluded in 1995 (4) that the uncertaintiesstill existed and confirmed the NOAEL for cyclohexylamine in the rat of 100 mg/kg bw as the



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basis for the ADI and the temporary ADI of 0-11 mg/kg bw for cyclamate was maintained,

pending submission of further data as follows:

i) Given the consumption of cyclamate is likely to be on a regular basis among users, the

following information is required on conversion rates in humans, using reasonable

sample sizes, to be submitted within 2 years of publicising this opinion in the SCFminutes:

a) to establish what proportion of initially low/negligible converters can become high

converters over time with repeated exposure for a few weeks to cyclamate;b) for those who do convert, to establish within individuals the range of conversion

rates which occur over time following repeated exposure to cyclamate, paying

particular attention to inter-individual differences in the time course of induction

and the persistence and magnitude of the capacity to convert in high converters.

ii) If feasible, in vitro studies to compare the relative sensitivity of human, monkey and

rat testicular tissue to cyclohexylamine.

iii) The completed reports of three Spanish epidemiology studies.

In response to this request the petitioner submitted further information concerning conversion

rates in human converters and non-converters, a review of the potential for in vitro

investigation of the testicular toxicity of cyclohexylamine, and reports of the two Spanish

epidemiology studies comprising fertility of workers involved in cyclamate manufacture and

a case-control study on cyclamate intake and male fertility. A report on intake survey in

Catalonia was also submitted.

The Committee is also aware of a recent publication on long term toxicity and carcinogenicityof cyclamate in primates (5). The study was inconclusive and the Committee was unable to

use it for risk assessment.

Intake surveys

The amount and type of all foods, drinks and drugs consumed in a random sample of 2450

subjects of the Catalan population, aged 6-75 years, were obtained by two 24-hour recalls at

different seasons in 1992 (6). A food frequency questionnaire with 77 items was used and

cyclamate intake was calculated from the reported intakes of foods and the sweetener content,

obtained from the respective manufacturers. 18% of the population consumed cyclamate

(21% in males, 15.4% in females), the highest percentage of 33% consumers was found inmen aged 35-44 years. Average daily cyclamate intake was estimated to be 0.4 and 2.4 mg/kgbw in the whole population and in consumers, respectively. In subjects on a diabetic diet (3%

of the population, no differentiation between consumers and non-consumers), the average

consumption of cyclamate was estimated to be 1.6 mg/kg bw. Among all consumers, the

highest intake of 4.9 and 4.4 mg/kg bw was estimated in males and females of the age group

6-17 years. Four subjects (0.16% of the population, 0.9% of consumers) were estimated to

have a cyclamate intake exceeding the tADI (i.e. >11 mg/kg bw).

In an earlier study performed in Germany (7), 31% of the study population (2291 persons)consumed cyclamate and the mean daily intake was estimated to be 2.6 and 6.1 mg/kg bw

among consumers and heavy consumers (90th

percentile of intake), respectively. Interestingly,persons who adhered to a diet (diabetes, weight control) did not ingest cyclamate in

substantially higher amounts. Similar figures for daily intake of cyclamate for users and



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persons on a diet were also reported from Brazil (8), while among 212 teenagers in Italy,

cyclamate use seems to be low (6% users with a mean daily intake of 0.24 mg/kg bw) (9). In

The Netherlands, 11% of the study population (6060 persons) consumed cyclamate and the

median daily intake estimated from a two-day record was 1.1 and 4.9 mg/kg bw among

consumers and heavy consumers (90th percentile of intake), respectively (10). Other intake

data on cyclamate were also available to the Committee. However these data were notincluded as intake estimates were not performed on a comparable basis.

Study on conversion rates in converters and non-converters.

In order to obtain a reasonable sample size of converters of cyclamate, an initial screening

study was performed with 261 subjects (125 males and 136 females) receiving the equivalent

of 3x250 mg cyclamic acid per day as calcium cyclamate during one week. Nine males and 10

females were identified as converters, based on urinary excretion of cyclohexylamine (>0.5%

metabolism of cyclamate to cyclohexylamine). 7 males and 7 females were selected and

agreed to participate in the main study as well as 31 non-converters (<0.3% metabolism, 19males and 12 females), based on urinary excretion of cyclohexylamine.

In the main study, the metabolism of cyclamate to cyclohexylamine was studied for 13 weeks

in the 45 subjects selected. The daily dose of cyclamic acid was again 3x250 mg, given in

tablets as calcium cyclamate. A controlled, timed 3-hour urine collection was performed twice

per week on weeks 1-3 and 7-13 and on the remaining study days, 20ml urine specimens

were collected. Blood samples were collected once weekly at the time of the 3-hour urine

collection. All samples were analyzed for cyclohexylamine.

Of the 31 non-converters, 30 essentially remained non-converters. One female subject showeda variable increase in the conversion rate, the highest rate was 1% on day 4. The average

conversion rate during steady state in this subject was 0.24%. Of the 14 subjects selected asconverters, one subject turned out to be essentially a non-converter (average conversion rate

during steady state 0.03%), 4 showed consistent low average conversion rate of 3-4% in the

steady state, 4 subjects showed an average conversion rate of 8-20% and 5 subjects converted

on the average between 25 and 46% of the cyclamate to cyclohexylamine. Large intra-

individual variations were observed in all converters. The highest conversions observed on

any single day in the 5 subjects with the highest average conversion rate range from 57-85%.

The individual variation can be illustrated by the highest converter (85,4% on day 60) who

converted 75% on day 49, 6.5% at day 56 and 42.7% at day 67; the average conversion rate

over days 7-91 was 26%. In 5 subjects, a tendency towards an increased conversion rate overtime was seen, reaching an average conversion rate during steady state of >18.9% and onesubject showing a constant average conversion rate of 36%. In the remaining 7 subjects

average conversion rate was <18.9% and no increase of the conversion rate over time was

observed. Plasma cyclohexylamine levels in converters were consistent with urinary data. A

high renal clearance of 447±127 ml/min (7.2±2.1 ml/min/kg bw) was found.

In conclusion, none of those initially non-converters became a high converter following

repeated exposure to cyclamate for 90 days. In the 13 converters studied, large intra-

individual daily variations were seen and in 6 subjects average conversion rate exceeded18.9% by a factor of up to 2.5.



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Discussion of in vitro studies to compare testicular sensitivity.

No in vitro studies to compare the relative sensitivity of human, monkey and rat testicular

tissue to cyclohexylamine were performed. Instead, a review of the available data in relation

to the potential use of in vitro testicular systems to study species sensitivity, as suggested by

the Committee, was submitted.

The testis is a complex tissue with many cell types and multiple interactions between the

various types. It is not possible to model the whole of the spermatic cycle by in vitro systems,limiting the value of testis-derived in vitro systems for investigating species comparisons of

toxicity, in the absence of knowledge on the target cell population, or the mechanism of

toxicity of a compound. The review concluded that target cells for testicular toxicity of

cyclohexylamine in the rat are most likely the Sertoli cells but target cells in dog and monkey

testes have not been identified. Therefore, an appropriate in vitro system should include both

(primary) Sertoli and germ cells. However, the relative in vitro/in vivo sensitivity of animal

species has not been established and no inter-laboratory validation studies have been

performed with such in vitro systems. Furthermore, no appropriate culture system forcomparing rat, monkey and human cells has been studied systematically. Although, in

principle, it could be possible to establish primary cell cultures of human Sertoli and germcells, the cells to be used are likely to be inappropriate for studying the sensitivity of the

developing testes to cyclohexylamine as the provenance of the cells would probably be from

elderly patients undergoing surgery due to prostatic cancer and healthy human tissue would

not be available.

In view of these major questions with respect to the reliability, reproducibility, and relevance

of any data on the sensitivity of animal and human testicular cells to cyclohexylamine, in vitro

studies for quantitative comparisons of the sensitivity of animal and human tissue wereconsidered to be not feasible.

Epidemiology studies: fertility of workers involved in cyclamate manufacture

A total of 18 workers involved in the manufacture of cyclamate were studied, 4 of them

having a high, direct exposure to cyclohexylamine for about 20 years and 1 had a high direct

exposure to cyclohexylamine for 2 years. Reproductive history, a semen sample and a 24-

hour urine specimen were provided by the workers. Because the workers were exposed to

other situations which could have affected fertility, such as elevated working temperature,

high alcohol consumption and smoking, the results are reported as a case study and nostatistical analysis could be performed. In the whole group, only 1 worker had a normal spermcount and normal sperm motility according to the WHO criteria (11). The 5 workers with high

direct cyclohexylamine exposure showed similar sperm counts, sperm motility and

reproductive performance to other workers without direct cyclohexylamine exposure.

Similarly, 10 workers with high measured urinary excretion of cyclohexylamine (0.12-1.45

mg/kg/day) were comparable to workers with a lower measured excretion of

cyclohexylamine. However, as the 18 workers form a seemingly abnormal group both with

respect to their low sperm count and their low sperm motility, the apparent lack of any

influence of cyclohexylamine in the 4 exposed workers on these parameters is difficult tointerpret and may not be extrapolated to the general healthy population. The Committee

concluded that this study is of little significance for the safety evaluation of cyclamate.



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Epidemiology studies: cyclamate intake and male fertility case control study.

Four hundred and five subjects, aged 30-50 years, attending a male infertility clinic in

Barcelona because of >12 months’ infertility were compared with 379 control subjects

referred to the same clinic for a vasectomy. Two semen samples and a 24-hour urine

specimen were provided by all subjects and the cyclamate intake was estimated from a semi-quantitative food frequency questionnaire between 1994 and 1996. There was no statistically

significant difference in the estimated cyclamate intake between cases (0.72 mg/kg bw/day)

and controls (0.55 mg/kg/day) or in the urinary cyclamate excretion (0.19 and 0.22 mg/kgbw/day in cases and controls, respectively). Cyclohexylamine was detected in the urine of

13% of cases and 12% of controls and the average values for urinary cyclohexylamine

excretion in cases and controls (0.035 and 0.053 mg/kg bw/day) were not statistically

significantly different.

Conclusion

The new epidemiological data revealed no indications of harmful effects on human

reproduction parameters of either cyclamate used as food additive or of workplace exposureto cyclohexylamine, though the latter study was considered difficult to interpret.

The Committee noted that no in vitro studies to compare the relative sensitivity of human,

monkey and rat testicular tissue to cyclohexylamine were performed but acknowledged the

difficulties in performing such studies. The Committee now no longer requires such studies.

After considering all the data available on conversion of cyclamate to cyclohexylamine in

humans, including the new data provided, the Committee concluded that the uncertaintieswith respect to the conversion rate in humans could be eliminated but that the 18.9%

conversion rate used for establishing the temporary ADI of 0-11 mg/kg bw is no longerappropriate. There are large inter-individual variations observed in conversion rates and a lack

of knowledge about the minimal time span of exposure to cyclohexylamine that might result

in testicular damage. The Committee therefore concluded that the maximum observed

individual overall conversion of cyclamate to cyclohexylamine and absorption of the latter

would be 85%. This would be more appropriate for calculating an ADI. Since a maximum

conversion figure would be utilised, a reduced safety factor should be applied for inter-

individual differences (see below).

The Committee concluded that a full ADI for cyclamate could now be established. Taking aNOAEL of 100 mg/kg bw for cyclohexylamine, allowing for the difference in molecularweight between cyclamic acid and cyclohexylamine, using an 85% overall conversion rate for

ingested cyclamate, and applying a 32-fold safety factor (see Annex), a full ADI of 0-7 mg/kg

bw, expressed as cyclamic acid, for cyclamic acid and its sodium and calcium salts was

established by the Committee.

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